Best practices of 2D and 3D CAR T cytotoxicity assay using high throughput plate based image cytometry

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. In the first part of this work we will expose the drawbacks of the the numerous traditional in vitro CAR-T cell-mediated cytotoxicity assays and demonstrate why image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance.
The success of CART has been limited to hematological blood born cancers and the transition into solid tumors has not been as effective as hoped. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment are but two methods that increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. In the second part of this work we will highlight why , with the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors.

Scott Cribbes

Senior Manager

Nexcelom from PerkinElmer

Biochemistry Honours Degree from Glasgow University followed by a PhD in Cancer biology from Beatson Institute for Cancer Research. Five years of research and assay development techniques at British Biotech, Axis Shield and AstraZeneca. Since 2001, I have worked at Applied Biosystems and Life Technologies in the area of imaging, HTS screening, and reagents solutions. Since 2009, I have worked with the Celigo image cytometer technology from development to released product for brightfield capability at Cyntellect to its current 5-channel format at Nexcelom, which continues development within the Perkin Elmer family. The Celigo image cytometer and Nexcelom’s family of cell counters seamlessly fits into the imaging portfolio of Perkin Elmer allowing us to provide scientist with innovative solutions for their research. I have now transitioned into the Market Development leader for Nexcelom products in EMEAI after running the EMEAI business as its development director for tteh last four years.

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Best practices of 2D and 3D CAR T cytotoxicity assay using high throughput plate based image cytometry
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Open to view video. In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. In the first part of this work we will expose the drawbacks of the the numerous traditional in vitro CAR-T cell-mediated cytotoxicity assays and demonstrate why image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. The success of CART has been limited to hematological blood born cancers and the transition into solid tumors has not been as effective as hoped. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment are but two methods that increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. In the second part of this work we will highlight why , with the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors.
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